Journal of Neurochemistry, 2004, 89, 1126–1136
doi:10.1111/j.1471-4159.2004.02378.x
Developmental loss and resistance to MPTP toxicity of dopaminergic neurones in substantia nigra pars compacta of c-synuclein, a-synuclein and double a/c-synuclein null mutant mice Darren C. Robertson,* Oliver Schmidt,* Natalia Ninkina,* Paul A. Jones, John Sharkey and Vladimir L. Buchman* *Department of Preclinical Veterinary Sciences and Fujisawa Institute of Neuroscience, University of Edinburgh, Edinburgh, UK
Abstract The growing body of evidence suggests that intermediate products of a-synuclein aggregation cause death of sensitive populations of neurones, particularly dopaminergic neurones, which is a critical event in the development of Parkinson’s disease and other synucleinopathies. The role of two other members of the family, b-synuclein and c-synuclein, in neurodegeneration is less understood. We studied the effect of inactivation of c-synuclein gene on mouse midbrain dopaminergic neurones. Reduced number of dopaminergic neurones was found in substantia nigra pars compacta (SNpc) but not in ventral tegmental area (VTA) of early post-natal and adult c-synuclein null mutant mice. Similar reductions were revealed in a-synuclein and double a-synuclein/c-synuclein
null mutant animals. However, in none of these mutants did this lead to significant changes of striatal dopamine or dopamine metabolite levels and motor dysfunction. In all three studied types of null mutants, dopaminergic neurones of SNpc were resistant to methyl-phenyl-tetrahydropyridine (MPTP) toxicity. We propose that both synucleins are important for effective survival of SNpc neurones during critical period of development but, in the absence of these proteins, permanent activation of compensatory mechanisms allow many neurones to survive and become resistant to certain toxic insults. Keywords: development, knockout mice, MPTP, substantia nigra, synuclein. J. Neurochem. (2004) 89, 1126–1136.
Discovery of two point mutations in the human a-synuclein gene associated with autosomal dominant familial form of Parkinson’s disease (Polymeropoulos et al. 1997; Kruger et al. 1998) triggered numerous studies aimed at understanding the mechanism of a-synuclein involvement in neurodegeneration. Although various observations suggested a causative role of a-synuclein aggregation in development of synucleinopathies (Spillantini et al. 1997, 1998a, 1998b; Conway et al. 1998, 2000; Irizarry et al. 1998; Mezey et al. 1998; Wakabayashi et al. 1998; Arai et al. 1999; Lippa et al. 1999; Narhi et al. 1999; Serpell et al. 2000; Takeda et al. 2000; Mori et al. 2002), it has also been shown that a-synuclein could cause cell death or render cells more sensitive to toxic insults independently of protein fibrillation (Ostrerova et al. 1999; Hsu et al. 2000; Saha et al. 2000; Lee et al. 2001; Gosavi et al. 2002; Lehmensiek et al. 2002; Petrucelli et al. 2002). This apparent discrepancy was at least partially explained when a multistep mechanism of
a-synuclein aggregation was revealed and the cytotoxicity was attributed to soluble a-synuclein oligomers or protofibrils, rather than insoluble highly polymerized mature fibrils (Wood et al. 1999; Stefanis et al. 2001; Volles et al. 2001; Ding et al. 2002; Gosavi et al. 2002; Lashuel et al. 2002; Volles and Lansbury 2002; Zhu et al. 2003).
1126
Received December 21, 2003; revised manuscript received January 10, 2004; accepted January 12, 2004. Address correspondence and reprint requests to Dr V. L. Buchman, Department of Preclinical Veterinary Sciences, University of Edinburgh, Summerhall, Edinburgh EH9 1QH, UK. E-mail:
[email protected] Abbreviations used: DA, dopamine; DAB, diaminobenzidine; DOPAC, 3,4-dihydroxyphenylacetic acid; FITC, fluorescein isothiocyanate; 5-HIAA, 5-hydroxyindolacetic acid; HPLC, high-performance liquid chromatography; HVA, homovanillic acid; MPTP, methyl-phenyl-tetrahydropyridine; NCD, natural cell death; PBS, phosphate-buffered saline; SDS–PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; SNpc, substantia nigra pars compacta; SNpc, substantia nigra pars compacta; TH, tyrosine hydroxylase; VTA, ventral tegmental area.
2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 1126–1136
Dopaminergic neurones in synuclein null mutant mice 1127
Overexpression of a-synuclein does not always have detrimental effects on the survival of cells in culture and, in some cells, has been shown to protect them from certain insults (Alves Da Costa et al. 2002; Lotharius et al. 2002; Wersinger and Sidhu 2003; Zourlidou et al. 2003). This suggests that the increase of intracellular concentration of a-synuclein is not the sole factor that changes cell fate; the presence and activity of certain macromolecules and/or intracellular structures and pathways, which interact either physically or functionally with a-synuclein are of no less importance. Among the factors shown to affect cell survival reciprocally with a-synuclein are free radical-producing metabolic pathways, excitotoxicity, signal transduction pathways, molecular chaperons, intracellular trafficking machinery, mitochondrial dysfunction and protein degradation systems, primarily proteasome (for recent reviews see Betarbet et al. 2002; Bonini 2002; Hardy 2002; Helfand 2002; Kruger et al. 2002; Lotharius and Brundin 2002; Mattson et al. 2002; Schwartz et al. 2002; Welch and Yuan 2002; Wolozin and Golts 2002; Cookson 2003; Dev et al. 2003; Lee 2003; Volles and Lansbury 2003). Recently, attention was focussed on the possible role of two other members of the family, b-synuclein and c-synuclein, in the regulation of a-synuclein-induced neurotoxicity. Although these two proteins are similar to a-synuclein in their amino acid sequences, they display rather different aggregation kinetics and do not form amyloid-like fibrils (reviewed in Uversky and Fink 2002). No mutations/ polymorphisms have been found so far in b-synuclein or c-synuclein genes in association with any neurodegenerative diseases (Lavedan et al. 1998b; Flowers et al. 1999; Lincoln et al. 1999a, 1999b; Kruger et al. 2001). Likewise, these two proteins have never been detected in histopathological hallmarks of neurodegeneration, although their presence in unusual structures or changes of their intracellular distribution have been reported in several cases of human neurological diseases (Duda et al. 1999; Galvin et al. 1999, 2000; Mori et al. 2002). Most interestingly, b-synuclein and c-synuclein inhibit the generation of a-synuclein protofibrils and fibrils in vitro (Uversky et al. 2002; Windisch et al. 2002; Park and Lansbury 2003), and are able to prevent neurotoxic effects of a-synuclein at least in certain in vivo systems (Hashimoto et al. 2001; Windisch et al. 2002; da Costa et al. 2003; and our unpublished observations). Taken together these suggest that correct balance of synucleins is important for normal brain function and imbalance of these proteins might affect survival of neurones that normally express more than one synuclein. However, recently we have shown that targeted inactivation of c-synuclein gene in mice does not affect survival of peripheral sensory and motor neurones that express high levels of this protein in wild-type animals (Ninkina et al. 2003). Nevertheless, because of compelling evidence that midbrain dopaminergic neurones are more vulnerable to changes of synuclein
metabolism than other neuronal populations (Zhou et al. 2000, 2002; Xu et al. 2002) it was expedient to study the effects of c-synuclein gene inactivation on these neurones. Here we demonstrate that in mouse SNpc, deficiency of c-synuclein, a-synuclein or both of these proteins, has negative effect on development of dopaminergic neurones, but renders them resistant to the toxic effect of MPTP.
Experimental procedures Materials Methyl-phenyl-tetrahydropyridine (MPTP), diaminobenzidine (DAB; Sigma Fast 3,3¢-diaminobenzidine tablet sets) and general chemicals were purchased from Sigma (St Louis, MO, USA), Vectastain ABC-kit from Vector Laboratories (Peterborough, UK), fluorescein isothiocyanate (FITC)-conjugated anti-mouse and TRITC-conjugated anti-rabbit immunoglobulins from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Generation of double c-synuclein/a-synuclein null-mutant mice Targeting inactivation of c-synuclein gene in ES cells and production of null mutant mice on pure C57Bl6J (Charles River Laboratories, Wilmington, MA, USA) background were described previously (Ninkina et al. 2003). A colony of a-synuclein mutant mice on pure genetic background was established from mice described previously (Abeliovich et al. 2000) by backcrossing with C57Bl6J mice (Charles River) for several generations as described elsewhere (Ninkina et al. 2003). Null mutant a-synuclein and c-synuclein mice were bred to produce double heterozygous and consequently double c-synuclein/a-synuclein null mutant mice. Immunoblotting Substantia nigra and surrounding midbrain region were dissected from adult mouse brains and homogenised in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) loading buffer followed by incubation of lysates for 5 min at 100C. To avoid the influence of individual differences and inconsistent dissections each lysate was prepared from tissues dissected from four brains. Samples of these lysates (10 lg of total protein) were analysed in 18% SDS–PAGE, transferred to Hybond-P membrane (Amersham, Arlington Heights, IL, USA) and probed with antibody as described previously (Buchman et al. 1998; Ninkina et al. 1999). Affinity-purified polyclonal rabbit SK23 antibody generated against C-terminal peptide of mouse c-synuclein (persyn; Buchman et al. 1998) was used in 1 : 500 dilution, sheep polyclonal antiasynuclein antibody (AB5334P, Chemicon, Temecula, CA, USA) – in 1 : 1000 dilution, rabbit polyclonal antib-synuclein antibody (AB5086, Chemicon) – in 1 : 3000 dilution and mouse monoclonal antia-tubulin antibody (DM1A, Sigma) – in 1 : 10000 dilution. Histology For the quantification of dopaminergic neurones in the midbrain of wild-type and mutant animals, brains were collected from E18 embryos, post-natal day 5 (P5) or adult mice. To minimize variations between the specimens, cohorts of brains from animals of the same age group were collected, processed, embedded and stained in parallel. Animals were killed by an appropriate Schedule 1 method
2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 1126–1136
1128 D. C. Robertson et al.
according to the UK Animals (Scientific Procedures) Act 1986. The brains were fixed in 4% paraformaldehyde/PBS (phosphate-buffered saline) or Carnoy’s fixative (60% ethanol, 30% chloroform, 10% glacial acetic acid) at 4C overnight following dehydration in alcohol series and embedding in paraffin blocks. Then, 8-lm-thick sections were cut using a HM 310 microtome (Microm International, Walldorf, Germany) and mounted onto Gold Seal slides (Gold Seal Products, Portsmouth, NH, USA). The paraffin sections were cleared in xylene and rehydrated through a graded alcohol series. Endogenous peroxidase activity was quenched by incubating the slides in 3% H2O2 in methanol for 20 min. After washing with PBS the tissues were blocked in 10% horse or goat serum and 0.4% Triton X-100 in PBS for 1 h at room temperature. Incubation with primary antibody was carried out at 4C overnight. A monoclonal antibody (clone 1B5) against tyrosine hydroxylase was purchased from Novocastra Laboratories (Newcastle upon Tyne, UK) and used in 1 : 40 dilution. Detection of immune complexes with biotinylated anti-mouse or anti-rabbit antibodies and avidin/peroxidase complex from Vectastain ABC-kit, and DAB as substrate were carried out according to manufacturers instructions. For analysis of c-synuclein protein expression and intracellular localization sections of the whole E15 embryos and E18, P2 or adult brains were prepared as described above and probed with 1 : 40 dilution of an SK23 antibody. For double immunoflourescent studies, FITC-conjugated anti-mouse and TRITC-conjugated anti-rabbit immunoglobulins were used in 1 : 200 dilution.
were expressed as mean ± SEM of seconds before fall for each experimental group.
Cell counts To assess number of dopaminergic neurones in SNpc and VTA regions of mouse brain, the first section for counting was randomly chosen from the first 10 sections that included SNpc/VTA region. Starting from this section, nuclei of tyrosine hydroxylase (TH)positive cells were counted on the every tenth section through the whole region. The Axiovision imaging program (Carl Zeiss Vision, Munchen-Hallbergmoss, Germany) was employed to measure diameters of nuclei of 50 randomly picked dopaminergic neurones in the SNpc or ventral ventral tegemental area (VTA) of every mouse brain included in this study. The average nuclear diameter for each structure in each brain was used for Abercrombie’s correction (Abercrombie 1946) of nuclei counts to obtain an actual number of TH positive cells in the structure. All counts were carried out blindly by a person unaware of the genotype of the animals.
Results
MPTP treatment Studies were approved by the Home Office and carried out according to the UK Animals (Scientific Procedures) Act 1986. All procedures, which included MPTP handling were carried out in accordance with published safety recommendations (Przedborski et al. 2001a). Eight- to 10-week-old wild-type or mutant male mice on C57Bl6 background were injected intrperitoneally (i.p.) with 0.1 mL of PBS or MPTP dissolved in PBS at 24-h intervals for 5 days. Daily dose of MPTP was 30 mg/kg. Brains were collected for histological studies 21 days after the last injection. Rotarod testing Seven-month-old male animals were tested thrice on a rotarod (UGO Basil, Comerio, Italy) at constant (24 rpm) rotation speed for 180 s or in accelerating (from 4 to 40 rpm) mode for 300 s. Results
Measurement of striatal dopamine and dopamine metabolite levels Brains of 9-month-old wild-type or mutant male mice on C57Bl6 background were dissected, the striatum removed on ice, snapfrozen and kept at )70C until assayed. 95 lL of 0.4 M HClO4 and 5 lL of 40 lg/mL N-x-5-HT (internal standard) was added to each thawed sample prior to sonication for 3 s by an ultrasonics homogenizer with a 3 mm tip. The samples were centrifuged at 20 000 g at a benchtop centrifuge for 25 min at 4C. The pellet was frozen for protein quantification, which was carried out using a standard BCA protein assay reagent kit (Pierce, Rockford, IL, USA). Then, 50 lL of the supernatant were injected onto the highperformance liquid chromatography (HPLC) column through a Rheodyne injection valve connected to a 20 lL loop. A BAS PM-80 solvent delivery system and BAS LC-4 ECD was used to detect dopamine (DA) and its metabolites (DOPAC, HVA and 5-HIAA). Isocratic mobile phase (75 mM sodium dihydrogen phosphate, 1.7 mM octanosulphonic acid sodium salt, 100 lL/L triethylamine in 90% milliQ water, 10% acetonitrile, pH 3.0) was used to carry the samples through a reverse phase ESA column (120 A C18 150 · 3.2 mm column packed with 3-lm particles). Flow rate was set at 0.6 mL/min and the detector was at 0.7 V.
Intracellular compartmentalisation of c-synuclein in substantia nigra neurones changes during development SK23 antibody, which specifically recognizes mouse c-synuclein was used to stain paraffin sections of embryonic and post-natal mouse brains. In agreement with previously published in situ hybridization data, expression of c-synuclein in neurones of subsatantia nigra or its primodium was detected at all developmental stages studied, although intracellular compartmentalisation of the protein was different at different stages. At E15, c-synuclein was detected in the primodium of substantia nigra both in neuronal cell bodies and in axons (Figs 1a and b). Later in embryonic and post-natal development, c-synuclein disappears from perikaria (Figs 1d and e), whereas axons of the nigro-striatal tract remain intensively stained (Figs 1c and g). However, in adult SNpc, haphazard TH-positive neurones display positive c-synuclein immunostaining in their cell bodies (Figs 1i and j). In developing and adult brain, punctuate neuropil staining was detected in striatum (data not shown), suggesting that c-synuclein is localized not only in axons but also in pre-synaptic regions of dopaminergic neurones of SNpc. Thus, neurones of the mouse nigro-striatal system display the same developmental dynamics of intracellular compartmentalization of c-synuclein as brain stem motoneurones (Ninkina et al. 2003), another neuronal population expressing high levels of this protein. Recently, we generated c-synuclein
2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 1126–1136
Dopaminergic neurones in synuclein null mutant mice 1129
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
Fig. 1 Intracellular comparetmentalisation of c-synuclein in developing and adult mouse nigro-striatal system. Immunostaining of paraffin sections of mouse E15 (a, b), E18 (c, d), P2 (e) or adult (g) brains with antic-synuclein antibody. In a sagittal section of E15 mouse brain (a), three small arrowheads denote the strongly stained fasciculus retroflexus, an important landmark in identifying the primordium of the substantia nigra (large arrowhead). An arrow designates the positively stained nigro-striatal tract. At a higher magnification (b), labelled cell bodies of the primordium of substantia nigra are visible. In a sagittal section of E18 mouse brain (c), the third and fourth ventricles are shown; other designations as in (a). At the higher magnification (d), no labelled cell bodies are visible but the primordial region is clearly outlined by a dotted neuropil stain. In a coronal section of P2 mouse brain (e), cell bodies of neurones in the red nucleus (RN) and nerve fibres in medial lemniscus (ML) are stained but no c-synuclein-positive cell bodies are apparent in VTA or SNpc. An adjacent section stained with anti-TH antibody to delineate VTA and SNpc is shown in (f). A fragment of sagittal section of adult mouse brain shows c-synucleinpositive axons in the nigro-striatal tract (g); an adjacent section stained with anti-TH antibody and counterstained with H&E (h). Doubleimmunoflourescent staining of adult SNpc neurones with antic-synuclein (i) and anti-TH (j) antibody. Scale bars are 500 lm (a and c), 20 lm (b) and 50 lm (d).
null mutant mice and demonstrated that the number of motoneurones in several brain stem nuclei is not affected by targeted inactivation of c-synuclein gene (Ninkina et al. 2003). However, various data suggest that dopaminergic neurones might be more susceptible to changes in
Fig. 2 The number of dopaminergic neurones in midbrain of wild-type and c-synuclein null mutant mice. Bar chart shows mean ± SEM of total number of TH-positive neurones in SN + VTA of E18, P5 and adult mice. No difference was found in E18 (p > 0.5, Student’s t-test; n ¼ 6 for each genotype) but P5 and adult c-synuclein null mutant mice (n ¼ 8 and 6, respectively) have significantly less (*p < 0.05, Student’s t-test) neurones than adult wild-type mice (n ¼ 9).
metabolism of synucleins than other types of neurones (Zhou et al. 2000, 2002; Kirik et al. 2002; Xu et al. 2002). Therefore, we studied the number of dopaminergic neurones in midbrain structures of c-synuclein null mutant mice. Deficit of dopaminergic neurones in substantia nigra pars compacta of c-synuclein null mutant mice In embryonic mouse brain, topographical discrimination between dopaminergic neurones of SN and VTA is problematic. Therefore, we compared the total (SN + VTA) numbers of TH-positive neurones in midbrain of wild-type and c-synuclein null mutant E18 embryos. At this developmental stage the same number of dopaminergic neurones was observed in SN + VTA of c-synuclein null and wild-type mice (Fig. 2). However, in early post-natal (P5) and adult (15–20 weeks) animals, small (15–20%) but statistically significant deficit of TH-positive neurones in SN + VTA of c-synuclein null mutant has been found (Fig. 2). To reveal if both structures lost the same proportion of neurones during post-natal development we studied more samples and counted separately the number of TH-positive neurones in SNpc and VTA of these two groups of adult mice. The results shown in Fig. 3 clearly demonstrate that the loss of dopaminergic neurones in c-synuclein null mutant mice takes place in SNpc (85.5 ± 5.2% of the number of neurones in wild-type mice) but not in VTA (102.5 ± 5.0% of the number of neurones in wild-type mice). In 18-month-old animals, the oldest studied so far, we found similar difference in the number of TH-positive neurones in SNpc between c-synuclein null mutant mice and wild-type littermates (81.4 ± 3.5% of the number of neurones in wild-type mice). Double c-synuclein/a-synuclein null mutant mice Inactivation of c-synuclein gene ultimately results in an increase of a-synuclein to c-synuclein ratio in cells that
2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 1126–1136
1130 D. C. Robertson et al.
Fig. 4 Performance of wild-type and synuclein null mutant mice in rotarod tests. Bar charts show mean ± SEM of time intervals from the test start to animal fall from the rotating rod. Results of 3 min test with constant, 24 rpm, rotation speed (a) and 5 min accelerating rotation test (b) are shown. Statistic analysis (Kruskal–Wallis one-way ANOVA) showed no significant difference in performance of wild-type and mutant mice in both tests (p > 0.4). Fig. 3 The number of dopaminergic neurones in midbrain structures of wild-type and synuclein null mutant mice. Bar charts show mean ± SEM of total number of TH-positive neurones in SNpc (a) or VTA (b) of wild-type (a+/+/c+/+), c-synuclein null mutant (a+/+/c–/–), a-synuclein null mutant (a–/–/c+/+) and double null mutant (a–/–/c–/–) mice. Neurones were counted separately in left and right structures of at least seven animals for each genotype. Statistic analysis (Kruskal– Wallis one-way ANOVA) showed no difference in VTA (p > 0.3) but significantly reduced number of neurones in SNpc (p < 0.01) of all three types of mutant mice when compared to wild-type mice.
normally express these two proteins, including dopaminergic neurones of SNpc. As such imbalance might be responsible for the increased toxicity of a-synuclein in the most vulnerable neuronal population, we produced mice lacking both a-synuclein as well as c-synuclein and studied neuronal complement in their SNpc and VTA. For these studies, a-synuclein-deficient mice on the pure C57Bl6J genetic background (Ninkina et al. 2003) were first crossed with c-synuclein-deficient mice on the same pure genetic background, to produce double heterozygous animals. Mice lacking both a-synuclein and c-synuclein genes were obtained in litters from double heterozygous parents with expected Mendelian frequency. Similar to both single null mutants, double null mutant mice were viable, fertile and did not show any obvious abnormalities in development, behaviour or in gross morphology of the nervous system (data not shown). Neither of our three populations of mutant mice developed motor dysfunction, as can be judged from their performance in either constant speed (Fig. 4a) or accelerated (Fig. 4b) rotarod tests. The number of TH-positive neurones in SNpc and VTA of adult double a-synuclein/c-synuclein null mutant mice
was similar (79.9 ± 5.3% and 91.4 ± 5.9% of the number of neurones in wild-type mice, respectively) to the number of TH-positive neurones in the same structures of c-synuclein null mutant mice (Fig. 3). Moreover, a similar deficit in the number of TH-positive neurones was found in SNpc (82.2 ± 4.4% of the number of neurones in wild-type mice) but not VTA (91.6 ± 5.3% of the number of neurones in wild-type mice) of a-synuclein null mutant mice (Fig. 3). To assess the effect of null mutation on the levels of dopamine and its metabolites, striatum was dissected from nine months old male mice and HPLC analysis was used to measure DA, DOPAC, 5-HIAA and HVA levels in the extract of each of individual striatum. For all four neurochemicals, no statistically significant differences were found between four studied genotypes (Fig. 5). Dopaminergic neurones of single and double synuclein deficient mice are resistant to MPTP toxicity To address the question of whether the absence of synucleins changes the sensitivity of dopaminergic neurones to specific toxic insults, we treated a-synuclein, c-synuclein and double a-synuclein/c-synuclein null mutant mice with MPTP, a neurotoxic drug that affects predominantly dopaminergic neurones of SNpc. A protocol of MPTP administration (see Experimental procedures) resulting in apoptotic death of dopaminergic neurones (Tatton and Kish 1997) has been chosen. This protocol allows for the identification both of animals more sensitive as well as less sensitive to the neurotoxic effect of the drug because it
2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 1126–1136
Dopaminergic neurones in synuclein null mutant mice 1131
Fig. 5 Dopamine and its metabolite levels in striatum of wild-type and synuclein null mutant mice. Striatal concentrations (ng/mg protein) of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindolacetic acid (5-HIAA) and homovanillic acid (HVA) in mutant animals were normalized to corresponding mean values for wild-type animals (100%) in each experiment. Mean ± SEM for nine animals per genotype from two separate experiments are shown. Statistical analysis showed no significant difference between wild-type and mutant mice for all four neurochemicals (p > 0.1, one-way ANOVA with posthoc Newman–Keuls test).
causes only moderate reduction in the number of THpositive neurones in SNpc of treated wild-type mice, which is not accompanied by substantial decrease of striatal dopamine level and, consequently, do not compromise animal performance in rotarod tests. As expected, in a group of wild-type mice this treatment substantially reduced the number of TH-positive neurones in SNpc (64.7 ± 11.1% of neurones remain in MPTP-treated group; 100% is the mean of neurone numbers in a vesicle-treated group of wild-type animals, p < 0.05) without significant effect on rotarod performance of these mice (Fig. 6). However, no significant decrease in number of THpositive neurones after MPTP treatment was observed in SNpc of mice lacking either a-synuclein (88.1 ± 4.7% of neurones remain in MPTP-treated group; 100% is the mean of neurone numbers in a vesicle-treated group of a-synuclein deficient animals, p > 0.1), or c-synuclein (95.3 ± 7.4% of neurones remain in MPTP-treated group; 100% is the mean of neurone numbers in a vesicle-treated group of c-synuclein deficient animals, p > 0.6), or both synucleins (85.9 ± 6.6% of neurones remain in MPTPtreated group; 100% is the mean of neurone numbers in a vesicle-treated group of double null mutant animals, p > 0.2; Fig. 6). Increased levels of b-synuclein in midbrain of single and double synuclein deficient mice Immunoblotting was used to compare levels of synucleins in midbrain of adult wild-type and synuclein null mutant mice. Equal amounts of total protein from four combined midbrains per each genotype were analysed using antibodies specific to a-, b- and c-synucleins and a-tubulin as a loading control. As expected, a-synuclein was absent in samples of a-synuclein null mutant and c-synucleins – in samples of c-synucleins null mutant animals, both these synucleins were
Fig. 6 Effect of chronic MPTP treatment on wild-type and synuclein null mutant mice. (a) Performance of wild-type (a+/c+), c-synuclein null mutant (a+/c–), a-synuclein null mutant (a–/c+) and double null mutant (a–/c–) mice in 5 min accelerating rotarod test before (–) and 2 weeks after (+) MPTP treatment. (b) The number of dopaminergic neurones in SNpc of wild-type and synuclein null mutant treated with MPTP (+) or vehicle (–). Neurones were counted separately in left and right SNpc of at least six animals for each experimental group. Statistic analysis (Kruskal–Wallis one-way ANOVA and Student’s t-test separately for each genotype) showed significant reduction in the number of neurones after MPTP treatment only for wild-type animals (*, see Results for details).
absent in samples of double null mutant animals (Fig. 7). In three independent experiments we consistently detected higher levels of b-synuclein in midbrain samples of all three null mutant mouse lines than in samples of wild-type mice (Fig. 7).
2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 1126–1136
1132 D. C. Robertson et al.
Fig. 7 Expression of synucleins in midbrain of adult mice. Each of three identical western blots with 10 lg of total protein lysates per lane were probed with antibody against a-synuclein, b-synuclein or c-synuclein. The filter probed with antib-synuclein antibody was re-probed with antia-tubulin antibody (lower panel). The lysates were prepared by homogenisation and boiling in SDS–PAGE loading buffer of midbrains of wild-type (a+/+/c+/+), c-synuclein (a+/+/c–/–), a-synuclein (a–/–/ c+/+) or double null mutant (a–/–/c–/–) mice (four combined midbrains for each genotype).
Discussion
In a search for phenotypical manifestations of targeted inactivation of c-synuclein gene in mice we carried out quantitative studies of mesencephalic dopaminergic neurones of SNpc and VTA, neuronal populations, which in wild-type adult mouse express high levels of c-synuclein mRNA (Lavedan et al. 1998a; Abeliovich et al. 2000; and our unpublished observations). A relatively small (15%) but consistent and statistically significant reduction in the number of TH-positive neurones has been found in SNpc of adult c-synuclein null mutant mice when compared with their wild-type littermates. This neuronal loss is not progressive, as the same difference in the number of neurones has been found between groups of ageing mutant and wild-type animals. Reduced number of midbrain dopaminergic neurones is already evident in P5 animals but earlier in development, at the late embryonic stage (E18), c-synuclein null mutant and wild-type mice have the same number of these neurones, suggesting that observed neuronal loss occurs peri- or early post-natally. It is well established that in rodents the natural cell death (NCD) of dopaminergic neurones in SNpc takes place during this perinatal and early post-natal period (reviewed in Burke 2003). Therefore it is feasible that the absence of c-synuclein makes dopaminergic neurones more vulnerable to NCD during this critical stage of their development. Despite the observed neuronal deficit, null mutant mice did not develop any detectable motor dysfunction. This is not surprising because clinical signs of nigro-striatal pathology become obvious only following a substantial decrease of striatal dopamine level, which in turn is usually associated with much more extensive loss of nigral neurones. Indeed, direct measurement of striatal dopamine and its metabolites has not shown significant difference in their levels between wild-type and null mutant animals. In
contrast to previously reported data (Abeliovich et al. 2000), we have not found reduction of striatal dopamine level in a-synuclein null mutant mice. The most probable explanation of this discrepancy is different genetic background of mice used in two studies. Abeliovich et al. (2000) used 129SV/j · C57Bl6 F2 intercrosses and all our experimental animals were transferred on pure C57Bl6 background as described earlier (Ninkina et al. 2003). Notably, two other groups that independently produced a-synuclein null mutant mice also did not find statistically significant decrease of their striatal dopamine levels (Cabin et al. 2002; Schluter et al. 2003). In the absence of a clear understanding of normal synuclein function, various scenarios can be employed to explain why c-synuclein null mutant mice develop a neuronal deficit in SNpc and why only a fraction of dopaminergic neurones are lost. Recently, the importance of a correct balance of synucleins for the survival of certain types of neurones has been proposed. Selective neurotoxicity of a-synuclein to dopaminergic neurones has been demonstrated in cell cultures (Zhou et al. 2000, 2002; Xu et al. 2002) as well as in invertebrate (Feany and Bender 2000; Auluck et al. 2002; Lakso et al. 2003), rodent (Kirik et al. 2002; Lo Bianco et al. 2002) and primate (Kirik et al. 2003) in vivo models. The ability of b-synuclein and c-synuclein to block formation of toxic intermediates of a-synuclein aggregation (Uversky et al. 2002; Windisch et al. 2002; Park and Lansbury 2003) and a-synuclein-induced cell death (Hashimoto et al. 2001; Windisch et al. 2002; our unpublished data) suggested that in the absence of endogenous b-synuclein or c-synuclein, dopaminergic neurones might become more susceptible to various internal and/or external insults due to the lack of natural counterbalance to the intrinsic toxicity of endogenous a-synuclein. However, our finding that dopaminergic neurones of VTA, which in wildtype mice express both a- and c-synuclein, are not affected in c-synuclein null mutant mice, suggests that neuronal loss seen in SNpc could not be explained only by selective toxicity of endogenous a-synuclein for dopaminergic neurones. Also, intracellular a-synuclein-positive inclusions, obvious indicators of the increased propensity of endogenous a-synuclein to aggregate in the absence of c-synuclein, have never been observed in brain sections of adult and ageing c-synuclein null mutant mice. Most importantly, we have found the same reduction in the number of TH-positive neurones in SNpc of double a-synuclein/c-synuclein null mutant mice as in c-synuclein null mutants. This suggests that the absence of a-synuclein does not rescue a susceptible population of c-synuclein-negative dopaminergic neurones from developmental cell death. Moreover, we have found similar neuronal deficit in SNpc of a-synuclein null mutant mice. The latter result is consistent with findings of another group, although authors did not comment on the significance of the difference they have shown (Dauer et al. 2002).
2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 1126–1136
Dopaminergic neurones in synuclein null mutant mice 1133
We believe that the most plausible interpretation of our experimental data is that both a- and c-synuclein are involved in and equally important for a certain intracellular process required for effective survival of SNpc dopaminergic neurones during a critical period of SNpc development. The roles of a- and c-synuclein in this process might be complementary, but not interchangeable. Therefore, the same neuronal population is affected in a-synuclein and c-synuclein null mutant mice and the absence of both these synucleins does not have an additive effect on neuronal survival. Experimental data obtained in various model systems implicate synucleins in many intracellular pathways associated with regulation of cell survival. The next obvious challenge is to reveal which of these pathways are indeed involved in synuclein-dependent survival of dopaminergic neurones of SNpc in the developing brain. However, it is obvious from our previous (Ninkina et al. 2003 and unpublished data) and present data, that in most neurones that normally express high levels of both a- and c-synuclein, including the majority of dopaminergic neurones of SNpc, the absence of these proteins are compensated and this allows them to survive through all critical stages of development. Furthermore, synuclein-deficient dopaminergic neurones of SNpc, which survived until adulthood, become resistant to MPTP toxicity. Stimulation of expression, post-translational modification and aggregation of a-synuclein in neurones of SNpc by MPTP are well documented, but it is less clear how these correlate with neuronal death (Kowall et al. 2000; Vila et al. 2000; Przedborski et al. 2001b; Meredith et al. 2002; Kuhn et al. 2003). Studies of dopaminergic neurones overexpressing different forms of human a-synuclein produced controversial results – either increased (Richfield et al. 2002) or unchanged (Rathke-Hartlieb et al. 2001; Dong et al. 2002) sensitivity to MPTP toxicity has been demonstrated. Results are more consistent for mice with targeted inactivation of a-synuclein gene. Resistance to MPTP toxicity has been shown previously for two independently generated mouse strains with targeted inactivation of a-synuclein gene (Dauer et al. 2002; Schluter et al. 2003) and we have demonstrated the same effect for the third strain. However, we have shown that dopaminergic neurones of SNpc of c-synuclein and double a-synuclein/c-synuclein null mutant mice are also resistant to MPTP toxicity. Concurrently, we detected increased levels of b-synuclein in midbrain of adult mice lacking a-, c- or both synucleins. Neuroprotective effect of b-synuclein overexpression has recently been demonstrated in different experimental systems (Hashimoto et al. 2001; da Costa et al. 2003) and it is feasible to suggest that compensatory increase of b-synuclein level in midbrain neurones survived beyond the period of NCD makes them less sensitive to certain neurotoxic insults. The levels of b-synuclein are similar in midbrains of a-synuclein and c-synuclein null mutant mice and loss of both a- and
c-synuclein in double mutant mice is not accompanied by further increase of b-synuclein level. These data are consistent with the discussed above idea that a- and c-synuclein are involved in the same intracellular processes and a suggestion that increased level of b-synuclein might be a factor required for effective survival of neurones in which these processes are compromised. Further experiments should reveal spatial and developmental patterns of b-synuclein accumulation in brains of mutant mice as well as if and how increased b-synuclein level is linked with activation of pro-survival or inhibition of pro-death mechanisms that might be responsible for resistance of synucleindeficient SNpc neurones to MPTP. Acknowledgements We are grateful to J. Wanless for excellent technical assistance. This work was supported by grants from The Wellcome Trust and a studentship to DR from MRC.
References Abeliovich A., Schmitz Y., Farinas I. et al. (2000) Mice lacking a-synuclein display functional deficits in the nigrostriatal dopamine system. Neuron 25, 239–252. Abercrombie M. (1946) Estimation of nuclear population from microtome sections. Anat. Rec 94, 239–247. Alves Da Costa C., Paitel E., Vincent B. and Checler F. (2002) a-Synuclein lowers p53-dependent apoptotic response of neuronal cells. Abolishment by 6-hydroxydopamine and implication for Parkinson’s disease. J. Biol. Chem. 277, 50980–50984. Arai T., Ueda K., Ikeda K. et al. (1999) Argyrophilic glial inclusions in the midbrain of patients with Parkinson’s disease and diffuse Lewy body disease are immunopositive for NACP/a-synuclein. Neurosci. Lett. 259, 83–86. Auluck P. K., Chan H. Y., Trojanowski J. Q., Lee V. M. and Bonini N. M. (2002) Chaperone suppression of a-synuclein toxicity in a Drosophila model for Parkinson’s disease. Science 295, 865–868. Betarbet R., Sherer T. B., Di Monte D. A. and Greenamyre J. T. (2002) Mechanistic approaches to Parkinson’s disease pathogenesis. Brain Pathol. 12, 499–510. Bonini N. M. (2002) Chaperoning brain degeneration. Proc. Natl Acad. Sci. USA 99, 16407–16411. Buchman V. L., Hunter H. J., Pinon L. G., Thompson J., Privalova E. M., Ninkina N. N. and Davies A. M. (1998) Persyn, a member of the synuclein family, has a distinct pattern of expression in the developing nervous system. J. Neurosci. 18, 9335–9341. Burke R. E. (2003) Post-natal developmental programmed cell death in dopamine neurons. Ann. NY Acad. Sci. 991, 69–79. Cabin D. E., Shimazu K., Murphy D. et al. (2002) Synaptic vesicle depletion correlates with attenuated synaptic responses to prolonged repetitive stimulation in mice lacking a-synuclein. J. Neurosci. 22, 8797–8807. Conway K. A., Harper J. D. and Lansbury P. T. (1998) Accelerated in vitro fibril formation by a mutant a-synuclein linked to earlyonset Parkinson disease. Nat. Med. 4, 1318–1320. Conway K. A., Harper J. D. and Lansbury P. T. Jr (2000) Fibrils formed in vitro from a-synuclein and two mutant forms linked to Parkinson’s disease are typical amyloid. Biochemistry 39, 2552–2563. Cookson M. R. (2003) Pathways to Parkinsonism. Neuron 37, 7–10.
2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 1126–1136
1134 D. C. Robertson et al.
da Costa C. A., Masliah E. and Checler F. (2003) b-Synuclein displays an antiapoptotic p53-dependent phenotype and protects neurons from 6-hydroxydopamine-induced caspase 3 activation: cross-talk with a-synuclein and implication for Parkinson’s disease. J. Biol. Chem. 278, 37330–37335. Dauer W., Kholodilov N., Vila M. et al. (2002) Resistance of a-synuclein null mice to the parkinsonian neurotoxin MPTP. Proc. Natl Acad. Sci. USA 99, 14524–14529. Dev K. K., Hofele K., Barbieri S., Buchman V. L. and van der Putten H. (2003) Part II: a-Synuclein and its molecular pathophysiological role in neurodegenerative disease. Neuropharmacology 45, 14–44. Ding T. T., Lee S. J., Rochet J. C. and Lansbury P. T. Jr (2002) Annular a-synuclein protofibrils are produced when spherical protofibrils are incubated in solution or bound to brain-derived membranes. Biochemistry 41, 10209–10217. Dong Z., Ferger B., Feldon J. and Bueler H. (2002) Overexpression of Parkinson’s disease-associated a-synucleinA53T by recombinant adeno-associated virus in mice does not increase the vulnerability of dopaminergic neurons to MPTP. J. Neurobiol. 53, 1–10. Duda J. E., Shah U., Arnold S. E., Lee V. M. and Trojanowski J. Q. (1999) The expression of a-, b-, and c-synucleins in olfactory mucosa from patients with and without neurodegenerative diseases. Exp. Neurol. 160, 515–522. Feany M. B. and Bender W. W. (2000) A Drosophila model of Parkinson’s disease. Nature 404, 394–398. Flowers J. M., Leigh P. N., Davies A. M., Ninkina N. N., Buchman V. L., Vaughan J., Wood N. W. and Powell J. F. (1999) Mutations in the gene encoding human persyn are not associated with amyotrophic lateral sclerosis or familial Parkinson’s disease. Neurosci. Lett. 274, 21–24. Galvin J. E., Uryu K., Lee V. M. and Trojanowski J. Q. (1999) Axon pathology in Parkinson’s disease and Lewy body dementia hippocampus contains a-, b-, and c-synuclein. Proc. Natl Acad. Sci. USA 96, 13450–13455. Galvin J. E., Giasson B., Hurtig H. I., Lee V. M. and Trojanowski J. Q. (2000) Neurodegeneration with brain iron accumulation, type 1 is characterized by a-, b-, and c-synuclein neuropathology. Am. J. Pathol. 157, 361–368. Gosavi N., Lee H. J., Lee J. S., Patel S. and Lee S. J. (2002) Golgi fragmentation occurs in the cells with pre-fibrillar a-synuclein aggregates and precedes the formation of fibrillar inclusion. J. Biol. Chem. 277, 48984–48992. Hardy J. (2002) Pathways to primary neurodegenerative disease. Neurologia 17, 399–401. Hashimoto M., Rockenstein E., Mante M., Mallory M. and Masliah E. (2001) b-Synuclein inhibits a-synuclein aggregation: a possible role as an anti-parkinsonian factor. Neuron 32, 213–223. Helfand S. L. (2002) Neurobiology: chaperones take flight. Science 295, 809–810. Hsu L. J., Sagara Y., Arroyo A. et al. (2000) a-Synuclein promotes mitochondrial deficit and oxidative stress. Am. J. Pathol. 157, 401– 410. Irizarry M. C., Growdon W., Gomez-Isla T., Newell K., George J. M., Clayton D. F. and Hyman B. T. (1998) Nigral and cortical Lewy bodies and dystrophic nigral neurites in Parkinson’s disease and cortical Lewy body disease contain a-synuclein immunoreactivity. J. Neuropathol. Exp. Neurol. 57, 334–337. Kirik D., Rosenblad C., Burger C., Lundberg C., Johansen T. E., Muzyczka N., Mandel R. J. and Bjorklund A. (2002) Parkinson-like neurodegeneration induced by targeted overexpression of a-synuclein in the nigrostriatal system. J. Neurosci. 22, 2780–2791. Kirik D., Annett L. E., Burger C., Muzyczka N., Mandel R. J. and Bjorklund A. (2003) Nigrostriatal a-synucleinopathy induced by viral vector-mediated overexpression of human a-synuclein: a new
primate model of Parkinson’s disease. Proc. Natl Acad. Sci. USA 100, 2884–2889. Kowall N. W., Hantraye P., Brouillet E., Beal M. F., McKee A. C. and Ferrante R. J. (2000) MPTP induces a-synuclein aggregation in the substantia nigra of baboons. Neuroreport 11, 211–213. Kruger R., Eberhardt O., Riess O. and Schulz J. B. (2002) Parkinson’s disease: one biochemical pathway to fit all genes? Trends Mol Med. 8, 236–240. Kruger R., Kuhn W., Muller T et al. (1998) Ala30Pro mutation in the gene encoding a-synuclein in Parkinson’s disease. Nat. Genet. 18, 106–108. Kruger R., Schols L., Muller T., Kuhn W., Woitalla D., Przuntek H., Epplen J. T. and Riess O. (2001) Evaluation of the c-synuclein gene in German Parkinson’s disease patients. Neurosci. Lett. 310, 191–193. Kuhn K., Wellen J., Link N., Maskri L., Lubbert H. and Stichel C. C. (2003) The mouse MPTP model: gene expression changes in dopaminergic neurons. Eur. J. Neurosci. 17, 1–12. Lakso M., Vartiainen S., Moilanen A. M., Sirvio J., Thomas J. H., Nass R., Blakely R. D. and Wong G. (2003) Dopaminergic neuronal loss and motor deficits in Caenorhabditis elegans overexpressing human a-synuclein. J. Neurochem. 86, 165–172. Lashuel H. A., Petre B. M., Wall J., Simon M., Nowak R. J., Walz T. and Lansbury P. T. Jr (2002) a-Synuclein, especially the Parkinson’s disease-associated mutants, forms pore-like annular and tubular protofibrils. J. Mol. Biol. 322, 1089–1102. Lavedan C., Buchholtz S., Auburger G. et al. (1998b) Absence of mutation in the b- and c-synuclein genes in familial autosomal dominant Parkinson’s disease. DNA Res. 5, 401–402. Lavedan C., Leroy E., Dehejia A., Buchholtz S., Dutra A., Nussbaum R. L. and Polymeropoulos M. H. (1998a) Identification, localization and characterization of the human c-synuclein gene. Hum. Genet. 103, 106–112. Lee S. J. (2003) a-Synuclein aggregation: a link between mitochondrial defects and Parkinson’s disease? Antioxid. Redox Signal 5, 337– 348. Lee M., Hyun D., Halliwell B. and Jenner P. (2001) Effect of the overexpression of wild-type or mutant a-synuclein on cell susceptibility to insult. J. Neurochem. 76, 998–1009. Lehmensiek V., Tan E. M., Schwarz J. and Storch A. (2002) Expression of mutant a-synucleins enhances dopamine transporter-mediated MPP+ toxicity in vitro. Neuroreport 13, 1279–1283. Lincoln S., Crook R., Chartier-Harlin M. C. et al. (1999a) No pathogenic mutations in the b-synuclein gene in Parkinson’s disease. Neurosci. Lett. 269, 107–109. Lincoln S., Gwinn-Hardy K., Goudreau J. et al. (1999b) No pathogenic mutations in the persyn gene in Parkinson’s disease. Neurosci. Lett. 259, 65–66. Lippa C. F., Schmidt M. L., Lee V. M. and Trojanowski J. Q. (1999) Antibodies to a-synuclein detect Lewy bodies in many Down’s syndrome brains with Alzheimer’s disease. Ann. Neurol. 45, 353–357. Lo Bianco C., Ridet J. L., Schneider B. L., Deglon N. and Aebischer P. (2002) a-Synucleinopathy and selective dopaminergic neuron loss in a rat lentiviral-based model of Parkinson’s disease. Proc. Natl Acad. Sci. USA 99, 10813–10818. Lotharius J., Barg S., Wiekop P., Lundberg C., Raymon H. K. and Brundin P. (2002) Effect of mutant a-synuclein on dopamine homeostasis in a new human mesencephalic cell line. J. Biol. Chem. 277, 38884–38894. Lotharius J. and Brundin P. (2002) Pathogenesis of Parkinson’s disease: dopamine, vesicles and a-synuclein. Nat. Rev. Neurosci. 3, 932– 942. Mattson M. P., Duan W., Chan S. L., Cheng A., Haughey N., Gary D. S., Guo Z., Lee J. and Furukawa K. (2002) Neuroprotective and
2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 1126–1136
Dopaminergic neurones in synuclein null mutant mice 1135
neurorestorative signal transduction mechanisms in brain aging: modification by genes, diet and behavior. Neurobiol. Aging 23, 695–705. Meredith G. E., Totterdell S., Petroske E., Santa Cruz K., Callison R. C. Jr and Lau Y. S. (2002) Lysosomal malfunction accompanies a-synuclein aggregation in a progressive mouse model of Parkinson’s disease. Brain Res. 956, 156–165. Mezey E., Dehejia A. M., Harta G., Tresser N., Suchy S. F., Nussbaum R. L., Brownstein M. J. and Polymeropoulos M. H. (1998) a-Synuclein is present in Lewy bodies in sporadic Parkinson’s disease. Mol. Psychiatry 3, 493–499. Mori F., Hayashi S., Yamagishi S., Yoshimoto M., Yagihashi S., Takahashi H. and Wakabayashi K. (2002) Pick’s disease: a- and b-synuclein-immunoreactive Pick bodies in the dentate gyrus. Acta Neuropathol. 104, 455–461. Narhi L., Wood S. J., Steavenson S. et al. (1999) Both familial Parkinson’s disease mutations accelerate a-synuclein aggregation. J. Biol. Chem. 274, 9843–9846. Ninkina N., Papachroni K., Robertson D. C. et al. (2003) Neurons expressing the highest levels of c-synuclein are unaffected by targeted inactivation of the gene. Mol. Cell Biol. 23, 8233–8245. Ninkina N. N., Privalova E. M., Pinon L. G., Davies A. M. and Buchman V. L. (1999) Developmentally regulated expression of persyn, a member of the synuclein family, in skin. Exp. Cell Res. 246, 308– 311. Ostrerova N., Petrucelli L., Farrer M., Mehta N., Choi P., Hardy J. and Wolozin B. (1999) a-Synuclein shares physical and functional homology with 14–3)3 proteins. J. Neurosci. 19, 5782–5791. Park J. Y. and Lansbury P. T. Jr (2003) b-Synuclein inhibits formation of a-synuclein protofibrils: a possible therapeutic strategy against Parkinson’s disease. Biochemistry 42, 3696–3700. Petrucelli L., O’Farrell C., Lockhart P. J. et al. (2002) Parkin protects against the toxicity associated with mutant a-synuclein: proteasome dysfunction selectively affects catecholaminergic neurons. Neuron 36, 1007–1019. Polymeropoulos M. H., Lavedan C., Leroy E. et al. (1997) Mutation in the a-synuclein gene identified in families with Parkinson’s disease. Science 276, 2045–2047. Przedborski S., Chen Q., Vila M., Giasson B. I., Djaldatti R., Vukosavic S., Souza J. M., Jackson-Lewis V., Lee V. M. and Ischiropoulos H. (2001b) Oxidative post-translational modifications of a-synuclein in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson’s disease. J. Neurochem. 76, 637–640. Przedborski S., Jackson-Lewis V., Naini A. B., Jakowec M., Petzinger G., Miller R. and Akram M. (2001a) The parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP): a technical review of its utility and safety. J. Neurochem. 76, 1265–1274. Rathke-Hartlieb S., Kahle P. J., Neumann M., Ozmen L., Haid S., Okochi M., Haass C. and Schulz J. B. (2001) Sensitivity to MPTP is not increased in Parkinson’s disease-associated mutant a-synuclein transgenic mice. J. Neurochem. 77, 1181–1184. Richfield E. K., Thiruchelvam M. J., Cory-Slechta D. A., Wuertzer C., Gainetdinov R. R., Caron M. G., Di Monte D. A. and Federoff H. J. (2002) Behavioral and neurochemical effects of wild-type and mutated human a-synuclein in transgenic mice. Exp. Neurol. 175, 35–48. Saha A. R., Ninkina N. N., Hanger D. P., Anderton B. H., Davies A. M. and Buchman V. L. (2000) Induction of neuronal death by a-synuclein. Eur. J. Neurosci. 12, 3073–3077. Schluter O. M., Fornai F., Alessandri M. G., Takamori S., Geppert M., Jahn R. and Sudhof T. C. (2003) Role of a-synuclein in 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine-induced parkinsonism in mice. Neuroscience 118, 985–1002.
Schwartz L. M., Nambu J. R. and Wang Z. (2002) Parkinsonism proteolysis and proteasomes. Cell Death Differ. 9, 479–482. Serpell L. C., Berriman J., Jakes R., Goedert M. and Crowther R. A. (2000) Fiber diffraction of synthetic a-synuclein filaments shows amyloid-like cross-b conformation. Proc. Natl Acad. Sci. USA 97, 4897–4902. Spillantini M. G., Schmidt M. L., Lee V. M., Trojanowski J. Q., Jakes R. and Goedert M. (1997) a-Synuclein in Lewy bodies. Nature 388, 839–840. Spillantini M. G., Crowther R. A., Jakes R., Cairns N. J., Lantos P. L. and Goedert M. (1998b) Filamentous a-synuclein inclusions link multiple system atrophy with Parkinson’s disease and dementia with Lewy bodies. Neurosci. Lett. 251, 205–208. Spillantini M. G., Crowther R. A., Jakes R., Hasegawa M. and Goedert M. (1998a) a-Synuclein in filamentous inclusions of Lewy bodies from Parkinson’s disease and dementia with lewy bodies. Proc. Natl Acad. Sci. USA 95, 6469–6473. Stefanis L., Larsen K. E., Rideout H. J., Sulzer D. and Greene L. A. (2001) Expression of A53T mutant but not wild-type a-synuclein in PC12 cells induces alterations of the ubiquitin-dependent degradation system, loss of dopamine release, and autophagic cell death. J. Neurosci. 21, 9549–9560. Takeda A., Hashimoto M., Mallory M., Sundsumo M., Hansen L. and Masliah E. (2000) C-terminal a-synuclein immunoreactivity in structures other than Lewy bodies in neurodegenerative disorders. Acta Neuropathol. 99, 296–304. Tatton N. A. and Kish S. J. (1997) In situ detection of apoptotic nuclei in the substantia nigra compacta of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice using terminal deoxynucleotidyl transferase labelling and acridine orange staining. Neuroscience 77, 1037–1048. Uversky V. N. and Fink A. L. (2002) Amino acid determinants of a-synuclein aggregation: putting together pieces of the puzzle. FEBS Lett. 522, 9–13. Uversky V. N., Li J., Souillac P., Millett I. S., Doniach S., Jakes R., Goedert M. and Fink A. L. (2002) Biophysical properties of the synucleins and their propensities to fibrillate: inhibition of a-synuclein assembly by b- and c-synucleins. J. Biol. Chem. 277, 11970–11978. Vila M., Vukosavic S., Jackson-Lewis V., Neystat M., Jakowec M. and Przedborski S. (2000) a-Synuclein upregulation in substantia nigra dopaminergic neurons following administration of the parkinsonian toxin MPTP. J. Neurochem. 74, 721–729. Volles M. J. and Lansbury P. T. Jr (2002) Vesicle permeabilization by protofibrillar a-synuclein is sensitive to Parkinson’s disease-linked mutations and occurs by a pore-like mechanism. Biochemistry 41, 4595–4602. Volles M. J. and Lansbury P. T. Jr (2003) Zeroing in on the pathogenic form of a-synuclein and its mechanism of neurotoxicity in Parkinson’s disease. Biochemistry 42, 7871–7878. Volles M. J., Lee S. J., Rochet J. C., Shtilerman M. D., Ding T. T., Kessler J. C. and Lansbury P. T. Jr (2001) Vesicle permeabilization by protofibrillar a-synuclein: implications for the pathogenesis and treatment of Parkinson’s disease. Biochemistry 40, 7812–7819. Wakabayashi K., Yoshimoto M., Tsuji S. and Takahashi H. (1998) a-Synuclein immunoreactivity in glial cytoplasmic inclusions in multiple system atrophy. Neurosci. Lett. 249, 180–182. Welch K. and Yuan J. (2002) Releasing the nerve cell killers. Nat. Med. 8, 564–565. Wersinger C. and Sidhu A. (2003) Differential cytotoxicity of dopamine and H2O2 in a human neuroblastoma divided cell line transfected with a-synuclein and its familial Parkinson’s disease-linked mutants. Neurosci. Lett. 342, 124–128.
2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 1126–1136
1136 D. C. Robertson et al.
Windisch M., Hutter-Paier B., Rockenstein E., Hashimoto M., Mallory M. and Masliah E. (2002) Development of a new treatment for Alzheimer’s disease and Parkinson’s disease using anti-aggregatory b-synuclein-derived peptides. J. Mol. Neurosci. 19, 63–69. Wolozin B. and Golts N. (2002) Iron and Parkinson’s disease. Neuroscientist 8, 22–32. Wood S. J., Wypych J., Steavenson S., Louis J. C., Citron M. and Biere A. L. (1999) a-Synuclein fibrillogenesis is nucleation-dependent. Implications for the pathogenesis of Parkinson’s disease. J. Biol. Chem. 274, 19509–19512. Xu J., Kao S. Y., Lee F. J., Song W., Jin L. W. and Yankner B. A. (2002) Dopamine-dependent neurotoxicity of a-synuclein: a mechanism for selective neurodegeneration in Parkinson’s disease. Nat. Med. 8, 600–606.
Zhou W., Hurlbert M. S., Schaack J., Prasad K. N. and Freed C. R. (2000) Overexpression of human a-synuclein causes dopamine neuron death in rat primary culture and immortalized mesencephalon-derived cells. Brain Res. 866, 33–43. Zhou W., Schaack J., Zawada W. M. and Freed C. R. (2002) Overexpression of human a-synuclein causes dopamine neuron death in primary human mesencephalic culture. Brain Res. 926, 42–50. Zhu M., Li J. and Fink A. L. (2003) The association of a-synuclein with membranes affects bilayer structure, stability and fibril formation. J. Biol. Chem. 278, 40186–40197. Zourlidou A., Payne Smith M. D. and Latchman D. S. (2003) Modulation of cell death by a-synuclein is stimulus-dependent in mammalian cells. Neurosci. Lett. 340, 234–238.
2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 1126–1136
